Ebola virus, EBOV, belongs to filoviridae, and can cause hemorrhagic fever of human beings and non-human primates. Ebola virus was discovered in Southern Sudan and the Ebola River areas of Zaire in 1976 for the first time, and the virus was named as “Ebola” hence. A patient suffering from the virus has symptoms of fever, emesis, diarrhea, external and internal bleeding, brain, liver and kidney injury and the like, the mortality rate is as high as 50-90%. Diseases caused by Ebola virus infection were called as “Ebola hemorrhagic fever” before 2014, and after the Ebola epidemic outbreak in West Africa in 2014, the WHO and the US CDC changed the name of Ebola hemorrhagic fever to Ebola virus diseases, EVD.
At present, there are 5 subspecies of Ebola viruses: Zaire ebolavirus (EBOV), Sudan ebolavirus (SUDV), TaiForest ebolavirus (TAFV, which is also named as Cote d'Ivoire or Ivory Coast), Bundibugyoe ebolavirus (BDBV) and Reston ebolavirus (RESTV). There were reports that human beings were infected by the first four Ebola viruses and similar clinical symptoms were caused, and the diseases only happened in Africa at present. The Zaire ebolavirus (EBOV) has the strongest pathogenicity, and the Sudan ebolavirus (SUDV) has the second strongest pathogenicity.
Before the serious Ebola epidemic in West Africa in 2014, there were 24 Ebola epidemic events in the history, the death toll was about 1500 in all, the epidemic events mainly happened in Congo, Uganda, Sudan and the like in middle Africa, the outbreak generally occurred in sparsely populated areas, and the death toll of a single epidemic outbreak was never greater than 300. In 2014, the epidemic started from Guinea, and rapidly spread to countries around such as Liberia and Sierra Leone, a great number of EVD patients appeared in densely populated cities such as the capital Conakry of Guinea, the capital Freetown of Sierra Leone, the capital Monrovia of Liberia and the capital Lagos of Nigeria, and the virus had the characteristic of being propagated along traffic trunks, and was propagated to other areas out of the African continent for the first time, such as America, England, Spain and the like. The death toll has been approximate to 11 thousand in 2014-2015 Ebola epidemic
There was no approved Ebola vaccine or treatment medicine in the world till March 2017. Many labs and pharmaceuticals companies are developing EBOV vaccines and treatment medicines. The vaccines include DNA vaccines, sub-unit vaccines, non-replication virus vector vaccines and replicable virus vector vaccines, and some DNA vaccines or vaccines based on live virus vectors already enter into clinical trials.
Ebola virus envelop Glycoprotein (GP) is the sole surface protein of an Ebola virus envelop, it has 2 reading frames, encoding a secretion small protein (sGP) and a whole-length transmembrane GP, respectively. GP is taken as a significant decisive factor of pathogenicity of the Ebola virus, and mediates virus enter the host cell by binding to a receptor, and is capable of damaging completeness of microvessels by binding to endothelial cells, and vascular leakage can be caused. At the same time, GP is also the main target protein inducing protective immune response. At present, all studies about the Ebola virus vaccines take GP (or GP and NP) as target antigen, and monoclonal antibodies (or antibody mixtures) which are proved to be effective in animal tests are all antibodies targeted to GP. Conventional inactivated vaccines and sub-unit vaccines are all ineffective on the Ebola virus, and vaccines which are being studied and are good in animal protection effect are ones which take GP as the target antigen and are based on virus vectors. How to obtain GP antigen as much as possible with a lowest immune dosage becomes an issue which needs to be solved urgently in development of Ebola vaccine.
An AdMax system consists of a shuttle plasmid with loxP site, a backbone plasmid and a HEK-293 cell strain, and after foreign gene sequences are constructed into the shuttle plasmid, the recombinant adenovirus can be generated through gene recombination with the backbone plasmid in HEK-293 cells.
The AdMax adenovirus system is a set of recombinant adenovirus construction system constructed by Frank Graham in 1999, and is developed as an adenovirus vector packaging system by the Micobix Company in Canada. The basic principle of the AdMax system for recombinant adenovirus packaging is that with Cre-loxP or FLP-frt recombinant enzymes, the shuttle plasmids and the backbone plasmids which are co-transfected into the HEK293 cells are subjected to site-specific recombination, generating the recombinant adenovirus. The recombinant virus obtained in such a manner is a replication defective with E1 loss, and the virus can only achieve foreign gene expression in cells without E1 area, and has no propagation ability. The AdMax system has the characteristic that recombinant virus generation is completed in a eukaryotic cell through the recombinant enzyme, is efficient and stable, and is one of the most convenient and rapidest adenovirus packaging systems at present.
Compared with most common AdEasy system at present, the AdMax system is relatively rapid in virus generation, and is capable of efficiently, simply, conveniently and rapidly acquiring the recombinant adenovirus, and moreover the yielding of the virus is greatly increased. By using the AdMax system, the whole process of plasmid construction to recombinant virus generation can be completed within only 2-4 weeks, the success rate is greater than 98% (95% of clone comprises target genes); since the recombinant virus generation is completed in the eukaryotic cell, the survival pressure to the adenovirus is maintained, and thus the completeness of a recombinant adenovirus genome can be maintained; however, the virus generation success rate of the AdEasy system is only 18-34% (the success rate of a new AdEasyXL system of the Stratagene company is about 94%), and in addition, because virus genome recombination is completed in a prokaryotic cell (BJ5183), theoretically, the adenovirus genome without the survival pressure can be relatively liable to mutate, and the virus inheritance background and the activity can be possibly affected.
Although the AdMax system has the advantages of being rapid in virus production, high in yielding and the like, as an effective live virus vector vaccine, in addition to advantages of the vector self, the system further needs to solve the significant issue how to more effectively express an inserted foreign target protein. If the foreign protein is too low in expression level, an effective immune effect is achieved by increasing the immune dosage, and the operation can definitely increase the adverse influence of the vector on inoculators. Therefore, optimizing the gene of the target protein so that the gene can be more effectively and correctly expressed, is an effective method for improving the immune effect of the vaccine.
On the basis of actual threat of the Ebola virus, the problem that in the prior art the vaccine prevention effect is not high in efficiency, and the technical difficulties of application of the AdMax system, the applicant intends to perform codon optimization on GP, so that the expression level of GP in the eukaryotic cell can be greatly increased, and the recombinant adenovirus vector Ebola vaccine which is relatively good in immunogenicity and capable of inducing antibodies of more high levels with a same dosage can be provided.